1. Field of the Invention
The invention relates to the field of chemical synthesis of oligonucleotides. More particularly, the invention relates to the synthesis of extremely high purity oligonucleotides.
2. Summary of the Related Art
Since the discovery by Zamecnik and Stephenson (Proc. Natl. Acad. Sci. 75, 280 (1978)) that synthetic oligonucleotides can inhibit Rous sarcoma virus replication, there has been great interest in the use of oligonucleotides and oligonucleotide analogs having modified internucleotide linkages to control gene regulation and to treat pathological conditions. There have been many reports of successful use of antisense oligonucleotides to inhibit gene expression both in vitro and in vivo, either directly by binding to double stranded DNA, or, primarily, indirectly by inhibiting translation of mRNA.
Many reports of successful antisense inhibition of nucleic acid expression in vitro have been reported. For example, Rapaport and Zamecnik (U.S. Pat. No. 5,616,564) disclosed successful antisense inhibition of malaria in parisitized erythrocytes. See also Barker et al. (Proc. Natl. Acad. Sci. USA 93, 514 (1996)). Oligodeoxyribonucleotide phosphorothioates have been found to inhibit immunodeficiency virus (Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079 (1988); Agrawal et al., Proc. Natl. Acad. Sci. USA 86, 7790 (1989); Agrawal et al., in Advanced Drug Delivery Reviews 6, 251 (R. Juliano, Ed., Elsevier, Amsterdam, 1991); Agrawal et al. in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, 143 (E. Wickstrom, Ed., Wiley/Liss, New York, 1991); and Zamecnik and Agrawal in Annual Review of AIDS Research, 301 (Koff et al., Eds., Dekker, New York, 1991)), and influenza virus (Leiter et al., Proc. Natl. Acad. Sci. USA 87, 3420-3434 (1990)) in tissue culture. In addition, oligodeoxyribonucleotide phosphorothioates have been the focus of a wide variety of basic research (e.g., Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401 (1990) and Eckstein and Gish, Trends Biochem. Sci. 14, 97 (1989)), enzyme inhibition studies (Mujumdar et al., Biochemistry 28, 1340 (1989)), regulation of oncogene expression (Reed et al., Cancer Res. 50, 6565 (1990)) and IL-1 expression (Manson et al., Lymphokine Res. 9, 35 (1990)) in tissue culture. A number of review articles report the many published studies of successful antisense inhibition in vitro. E.g., Uhlmann and Peyman, Chem. Rev. 90, 543 (1990).
A number of published reports disclose the successful antisense inhibition of nucleic acid expression in vivo. For example, Offensperger et al. (EMBO J. 12, 1257 (1993)) demonstrated in vivo inhibition of duck hepatitis B virus. Nesterova and Cho-Chung (Nat. Med. 1, 528 (1995)) demonstrated inhibition of tumor growth by a single subcutaneous injection of antisense phosphorothioate oligonucleotide targeted to the RI, subunit of protein kinase A in nude mice. Several general reviews of in vivo antisense inhibition have appeared that discuss these and other studies demonstrating successful in vivo antisense inhibition of nucleic acid expression as well as applications for therapeutic use. See, e.g., Agrawal, TIBTECH 14, 376 (1996); Field and Goodchild, J. Exp. Opin. Invest. Drugs 4, 799 (1995).
These and other studies have proven sufficiently successful to justify extension to humans. A number of human clinical trials are currently ongoing, testing antisense oligonucleotides against a variety of disease causing targets, including HIV, CMV retinitis, ICAM, PKC, c-myb, and c-raf.
A necessary precursor to using antisense oligonucleotides to inhibit nucleic acid expression is the synthesis of the oligonucleotides. Various methods have been developed for the synthesis of oligonucleotides for such purposes. Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al. (J. Molec. Biol. 72, 209 (1972)) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese (Tetrahedron Lett. 34, 3143 (1978)) discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers (Tetrahedron Lett. 22, 1859 (1981)) discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik (U.S. Pat. No. 5,149,798) discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild (Tetrahedron Lett. 28, 3539 (1987)) teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al. (Biochemistry 23, 3443 (1984)) discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al. (Biochemistry 27, 7237 (1988)) discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al. (Proc. Natl. Acad. Sci. USA 85, 7079 (1988)) discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
A number of treatises and review articles have appeared that discuss the various synthetic approaches. E.g., Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, p. 63-80 (S. Agrawal, Ed., Humana Press 1993); Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates (Agrawal, Ed., Humana Press, Totowa, NJ 1994); Oligonucleotides and Analogues: A Practical Approach pp. 155-183 (Eckstein, Ed., IRL Press, Oxford 1991); Antisense Res. and Applns. pp. 375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, FL 1993); Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds., Raven Press, New York, 1992).
Both phosphoramidite and H-phosphonate chemical syntheses are carried out on a solid support that is stored in a reaction vessel. The required reaction steps for coupling each nucleotide are detritylation, coupling, capping, and oxidation. For small scale (up to 1 .mu.mole) synthesis, the total time for the addition of one nucleotide is about 6 minutes. An oligonucleotide, 30-mer in length, can be assembled in 180 minutes. Under these conditions, synthesized oligonucleotides are chemically pure and biologically active. However, when oligonucleotides are synthesized on a larger scale (up to 1 mmole), the time for addition of each nucleotide onto CPG is in the range of 30 to 60 minutes, requiring approximately 12-25 hours for assembling a 25-mer oligonucleotide. The increase in time is due to the volume of the solid support being used in synthesis. This increase in cycle time exposes the already assembled oligonucleotide sequence to all reaction steps (including dichloroacetic acid detritylation step, coupling step, oxidation step and capping step) for a longer time. This increase in total assembly time affects the yield as well as chemical and biological properties of the compound. The chemical and biological properties are mainly affected by depurination, base modifications, and the like.
To reduce the effects of these problems, it is possible to synthesize oligonucleotides using dimeric or multimeric synthons, thereby reducing the number of cycles, and thus the time required for synthesizing oligonucleotides. To this end, several investigators have worked toward developing acceptable dimeric or multimeric synthon approaches. Khorana (Science 203, 614 (1979)) introduced the concept of multimeric synthons, using a phosphodiester approach. Crea and Itakura (Proc. Natl. Acad. Sci. USA 75, 5765 (1978)), Reese (Tetrahedron Lett. 34, 3143 (1978)), and Ohtsuka et al. (Nucleic Acids Res. 10, 6553 (1982)) all disclose use of dimeric or multimeric synthons in a phosphotriester approach. Kumar and Poonian (J. Org. Chem. 49, 4905 (1984)) and Wolter et al. (Nucleosides and Nucleotides 5, 65 (1986)) disclose synthesis of oligonucleotide phosphodiesters using dimeric phosphoramidite synthons. Marugg et al. (Nucleic Acids Res. 12, 9095 (1984)) teaches use of a dinucleotide thiophosphotriester to produce oligonucleotides containing one phosphorothioate linkage. Connolly et al. (Biochemistry 23, 3443 (1984)) and Cosstick and Eckstein (Biochemistry 24, 3630 (1985)) disclose addition of one dinucleotide phosphorothioate to a growing oligonucleotide chain using a phosphoramidite approach. Brill and Caruthers (Tetrahedron Lett. 28, 3205 (1987)) discloses synthesis of thymidine dinucleotide methylphosphonothioates. Roelen et al. (Nucleic Acids Res. 16, 7633 (1988)) discloses a solution phase approach, using a reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethyl benzotriazole to introduce a methylphosphonothioate internucleotide linkage into a dinucleotide in 60-70% yield, and produces a hexamer containing the linkage by two consecutive condensations of dimers. Roelen et al. (Tetrahedron Lett. 33, 2357 (1992)), discloses reagents for alkylphosphonate and alkylphosphonothioate chemistry. It discloses the solution phase synthesis of TG methyl, n-butyl, and n-octyl phosphonate and phosphonothioate dimers.
Leba dev et al. (Tetrahedron. Lett. 31, 855 (1990)) discloses a solution phase approach to produce dinucleotides containing a stereospecific methylphosphonothioate internucleotide linkage in 50-60% yield. Katti and Agarwal (Tetrahedron Lett. 27, 5327 (1986)) discloses 3' 1-methoxycarbonate methylphosphonate dimers.
The use of such synthons in the synthesis of oligonucleotides has also been disclosed. Kumar and Poonian, supra, demonstrated the use of 3' phosphoramidite methyl phosphotriester dimers in the solid phase manual synthesis of a 29-mer with an overall yield of 93.4%. Wolter, supra, demonstrated the automated, solid phase synthesis of a 101-mer using .beta.-cyanoethyl-protected phosphoramidite dimers. Miura et al. (Chem. Pharm. Bull. 35, 833 (1987)) discloses automated solid-phase synthesis of pentadecathymidilate with phosphoramidite dimers. And Bannwarth (Helv. Chim. Acta 68, 1907 (1985) disclosed the use of phosphoramidite dinucleotides in the synthesis of oligonucleotides of modest length (N=8-11).
Krotz et al. recently reported the synthesis of phosphorothioate dimers having low phosphodiester dimer content. They used these dimers to synthesize phosphorothioate oligo(T) and oligo(TC) nucleotides, wherein they observed that the N/N-1 ratio was on the order of 99:1 as measured by capillary gel electrophoresis (CGE). The phosphodiester content of the oligomers was on the order of 1% as determined by .sup.31 P NMR for oligomers synthesized with phosphorothioate dimers wherein the phosphorothioate linkage is protected by a .beta.-cyanoethyl group on the non-bridging oxygen. A similar reduction of the phosphodiester content was not observed for dimers wherein the phosphorothioate linkage was protected by a .beta.-cyanoethyl group on the (non-linking) sulfur.
Once synthesized, the desired oligonucleotide (being "N" nucleotides in length) must be isolated from failure sequences (i.e., sequences with fewer than "N" nucleotides, such as N-1, N-2, etc.) and other impurities. While automated synthesizers have proven an invaluable tool for obtaining oligonucleotides, 1-3% of the reactions fail during each cycle in which a nucleotide monomer is to be added. Consequently, the resulting products are generally a heterogenous mixture of oligonucleotides of varying length. For example, in a typical 20mer synthesis, the 20mer product represents only 50-80% of the recovered oligonucleotide product.
Furthermore, preparation of oligodeoxynucleotides on a solid phase support requires that the oligodeoxynucleotide be cleaved from the support. Cleavage of the oligonucleotide from the support is typically accomplished by treating the solid phase with concentrated ammonium hydroxide. The ammonium hydroxide is conventionally removed under reduced pressure using, for example, a rotary evaporator. This method for removing the ammonium hydroxide, however, is not ideal for use in large scale isolation of oligodeoxynucleotides.
For most purposes (e.g., therapeutic or diagnostic) the purity of the compounds is extremely important. Consequently, there has been an interest in developing chromatographic techniques for purifying oligonucleotides. Because of their therapeutic potential, much of the focus has been on purifying oligonucleotide phosphorothioates.
Conventional methods for purifying oligodeoxynucleotides employ reverse-phase liquid chromatography. Manufacturing facilities using such methods require explosion-proof equipment because acetonitrile is typically used in the elution buffer.
Methods of oligodeoxynucleotide phosphorothioate purification have been published. Metelev and Agrawal (Anal. Biochem. 200, 342 (1992)) reported the ion-exchange HPLC analysis of oligodeoxyribonucleotide phosphorothioates on a weak anion-exchange column (Partisphere WAX) in which the weak anion exchanger utilizes a dimethylaminopropyl functional group bonded to Partisphere silica. This medium, with an ion-exchange capacity of 0.18 meq/g, exhibits an interaction with anions weaker than those observed with strong anion-exchange media. The authors of this study found that separation was length dependent for oligonucleotide phosphorothioates up to 25 nucleotides in length. Furthermore, N-1 peaks were separated from the parent peak. They also found that 30-mer and 35-mer oligonucleotide phosphorothioates were separable with the same gradient, although better separation could be obtained with a shallower gradient.
Metelev et al. (Ann. N.Y Acad. Sci. 660, 321-323 (1992)) reported the analysis of oligoribonucleotides and chimeric oligoribo-oligodeoxyribonucleotides using ion-exchange HPLC. They found that the retention time of the oligonucleotides studied depended on the number of ribonucleotide moieties in the oligonucleotide. In addition, the retention time of oligoribonucleotides was found to be length dependent. The authors noted that oligoribonucleotides of length up to 25 nucleotides could be purified and analyzed.
Bigelow et al. (J. Chromatography 533, 131 (1990)) reported the use of ion-pair HPLC to analyze oligonucleotide phosphorothioates. Stec. et al. (J. Chromatography 326, 263 (1985)) and Agrawal and Zamecnik (Nucleic Acids Res. 19, 5419 (1990)), reported HPLC analysis of oligodeoxyribonucleotides containing one or two phosphorothioate internucleotide linkages using a reversed-phase column.
Tang et al. (WO 95/27718) disclosed a purification techniques suitable for large scale separation of oligonucleotide phosphorothioates. The method uses DMAE Fractogel EMD column with an organic solvent-free, low salt, elution buffer. The method does not require elevated temperatures, making it more amenable for large scale chromatography.
Puma et al. (WO 96/01268) disclosed a purification method not requiring the removal of ammonium hydroxide or the use of conventional C-18 silica gel reverse-phase liquid chromatography. The disclosed methods use hydrophobic interaction chromatography and DEAE-5PW anion ion-exchange chromatography.
As antisense oligonucleotides proceed through human clinical trials, there is an ever-increasing demand for extremely pure oligonucleotides in large quantities. Regulatory agencies around the world are addressing the requisite standards for antisense oligonucleotides as drug compounds. E.g., Kambhampati et al, Antisense Res. Dev. 3, 405 (1993). Consequently, there remains a need for new methods of producing large quantities of highly pure oligonucleotides.